The overall goal of this research project is to understand the molecular mechanisms which regulate aflatoxin B1 biosynthesis in Aspergillus parasiticus. The working hypothesis is that aflatoxin B1 biosynthesis is regulated at the transcriptional level. The following specific aims are proposed to test this hypothesis: (1) Clone genes encoding enzymes which catalyze aflatoxin biosynthesis by genetic complementation of A. parasiticus mutants ("blocked mutants") deficient in unique enzymatic steps in the aflatoxin biosynthetic pathway by transformation with a cosmid library; and (2) characterize the genetic organization and expression of the cloned genes by: a) nucleotide sequence analysis, b) Southern and Northern analysis, c) S-1 nuclease analysis, and d) transverse alternating field gel electrophoresis (TAFE). It is hoped that elucidation of the mechanisms controlling this anabolic pathway will provide targets for inhibition of aflatoxin biosynthesis by alternation of the regulatory scheme. In the long term, this may lead to prevention of preharvest aflatoxigenesis by application of biological/chemical inhibitors of this pathway, competitive exclusion by nonaflatoxigenic aspergilli, or genetic modification of host plants. Since transcriptional regulation of secondary metabolites may involve common regulatory elements among different eukaryotic genera, the aflatoxin pathway may provide a model for also understanding the biosynthesis of other mycotoxins and toxic secondary metabolites that impact public health and food safety.